All About Cell Culture

This is it! The complete guide to performing cell culture!

Whether you are new to working with mammalian cells or you have wealth of experience with cells and just want to compare methods, you are so welcome to keep on reading. Cell culture is done in a very straightforward principle. When we grow them in the lab, most of the time it is on 2D flat surface of culture plates, which represent an in vitro setting.  This serves as a platform for having a miniaturized biological system where we can subject the cells to many treatments, chemically and physically. This leads to discovery of many characteristics of mammalian cells, their behavior when subjected to certain treatment. As the field progresses, more culture systems have been developed to somehow simulate a closer physiological environment for the cells, such as spheroid culture done in a round bottomed wells, and so on. Overall, these are still accounted as in vitro culture and they have been reliable in translating cell or tissue responses to treatments, and contributed to discoveries of therapies.

Terminology that you will encounter in cell culture:

- Basal medium = this usually contains the base medium, it can be Basal Eagle Medium (BME), Dulbecco's Modified Eagle Medium), or (RPMI), and there are plenty kinds of basal medium depending on the type of cells you grow. It is best to check with the cell bank, ATCC, or the supplier where you obtain the cell.

- Standard Culture Medium = this is usually made of basal medium, 1% penicillin -streptomycin, 1% L-glutamine, and 10% Fetal Bovine Serum (FBS)

- Confluent (adj) / confluency (noun) = the culture is confluent when cells have grown on or occupied all the available surface area due to proliferation

- Passaging = the activity of transferring cells from one flask/plate to another, typically when a culture has reached >90% confluency. It can also be referred as "Subculture".

- Passage number = the number of times the culture has been passaged. Many cells lose their proliferative and stemness behaviour after certain passage, so the earlier passage number, the better.

- Splitting = the activity of dividing a culture into multiple cultures. This shares the same idea with passaging. For example, from 1 confluent flask you may split it into 2, 3, 4.......and so on. as a result, each flask will have one half, one third, one fourth,...... of the original flask.

- The "hood" = which most of the time refers to the biosafety cabinet

- Seeding = placing cells in a well plate or flask at a certain density

- Contamination = when bacterial or fungal growth is found in the cell culture. But it can also mean when the growth of unwanted cells is found in a culture

- Cross contamination = when you are handling different cell lines at a time and it is possible that you mix them due to possible use of same pipette, same tips,

- Contact inhibition = it is the behavior of the cell that in the culture condition, they

- Adherent = Cells culture in a standard plastic culture flask will adhere to the surface after few hours of seeding

- Primary culture = The very first initiation of cell culture where tissue (taken from a donor or a animal) is digested and grown on the plates to allow cells to egress and grow further

- Cell line = the cells cultured after the successful initiation from primary culture

Prior to any cell culture procedure, make sure you do:

• prepare the workspace in the biosafety cabinet, make sure it has been UV decontaminated for at least 20 minutes and surface is wiped with 70% ethanol
• pre-warm the medium, 1X PBS and 0.25% trypsin-EDTA to be at 37 C
• thaw any frozen reagents that you might need (drugs or serum usually stored in -20 C)
• disinfect equipment you need in the hood by wiping them with ethanol such as sterile new plates or flask, pipettes, pipette gun, tubes.
• prepare sterile (autoclaved tips)
• label tubes , flask, plates needed during the procedure
• make sure the flow of the air filtration in the hood runs at a compatible rate and nothing obstruct this flow (height of glass sash, or things placed on the air intake grills)

More detailed steps:

Harvesting

It is the step of collecting cells for the purpose of passaging, seeding, or extracting constituents from cells.

1. To harvest cells in confluent T75 flask, retrieve cells from the incubator.
2. Remove the medium. Add 4 ml 1X PBS to wash. Rock the flask gently to evenly cover the surface if culture with PBS. This step can be done twice.
3. Remove PBS, add 3 ml of 0.25% Trypsin-EDTA
4. Close the flask and keep it in incubator for 5 min
5. After 5 mins, check for cell detachment under the microscope. Tap the side of the flask gently to help detach the rest of the cells.
6. As most cells detached, in the hood, add 7 ml standard culture medium to the flask. Make sure to blast off the surface of the flask as you aspirate and release the medium.
7. Transfer the whole cell suspension to a falcon tube
8. Centrifuge at 1500 rpm for 5 mins
9. There you have the cells collected. From this, cells can then be used further for passaging, seeding, etc.

Passaging

Passaging is the action of transferring cells that are growing rapidly in a flask or plate that confluency is reached. A 100% confluent culture flask means that the cells have grown on all the available surface area of the flask. Ideally, when confluency is about 80-90%, it means that passaging is required so that cells can have a new available surface to grow. Too confluent culture can result in cell death and loss of important function that may affect the result of your experiment.

1. To passage, following the steps above after centrifugation, remove the supernatant.
2. To the pellet, add 1 ml of medium first and re-suspend the pellet well by using the 1000 ul pipette
3. Then, top it up with 9 mL medium to make the total suspension volume 10 mL, mix well.
4. For passaging of ratio 1 : 10, take 1 mL from the suspension and add it to the new flask. This is taking 1/10 of the previous culture to the new one. This ratio depends on the confluency of the cell and the rate of cells to reach confluency. So it is best to really get to know this characteristic of your cells to time it well for your experiment.
5. In the new flask, top it up with 14 ml medium. The total volume in T 75 flask is 15 ml.
6. Keep the newly passaged cells in the incubator at 37C with 5% CO2

Counting

This is an important step to seed the cells at a desired number or density required for experiment. Counting using hemocytometer or the Neubauer's chamber is the most common procedure in cell culture.

1. From the harvesting steps above, after centrifugation, remove the supernatant.
2. To the pellet, add 1 ml of medium first and re-suspend the pellet well by using the 1000 ul pipette
3. Then, top it up with 9 mL medium, mix well by pipetting and invert the tube.
4. Take 15 ul from the suspension and transfer into a new eppendorf tube.
5. Take the tube to the nearest inverted microscope and add 15 ul of 0.4% Trypan blue to the eppendorf, mix well. This makes the dilution factor = 2
6. Take the 15 ul of the mixture and add to the haemocytometer with cover slip.
7. This is what should be seen under the microscope. You are only interested in counting the bright colored cells present in and on the boundary line of the 4x4 squares highlighted in red below. 
   
8. Using a counter, count carefully all the cells in all 4 quadrants. Take the average of the total number of cells in all these squares (divided by 4), then multiplied by 2 (dilution factor), then finally multiplied by 10,000. That will give you the number of cells per mL.
9. Make sure for this step, cell pellet is re-suspended very well to give a homogeneous solution. 

Seeding

1. With a known cell number, you can work out the volume needed to seed for your experiment.
2. For example, if you want to seed 10,000 cells per well in a 24 well plate (working volume is 1 ml per well), so the total number of cells you need is: 24 wells x 10,000 = 240,000 cells in 24 ml. If you have a confluent T75 flask (given 500,000 cells per ml).
3. Number of cells needed / number of cells given = volume you need to take
   240,000  / 500,000 = 0.48 ml = 480 ul
   To make 24 ml volume = 480 ul + 23.52 ml medium to add
   
   
4. Add 1 ml of this suspension to each well of the 24 well plate, and you get 10,000 cells per well

Freezing or Cryopreserving

1. Prior to freezing cell, prepare an esky with dry ice, or pre cool the Mr. Frosty / Freezing Jar containing isopropanol. Label all your cryovials with the name of cells, date, passage number, GMO details, and owner initials. The more detailed info you put, the more thankful you will be later on for keeping good record.

2. To freeze cells for future use, harvest cells following the steps above. After centrifugation, remove the supernatant.

3. From a  confluent T75 flask, you can usually split this to freeze into 3-4 vials. 1 vial contains 1 - 1.5 ml.

4. Prepare freezing medium, which contains standard culture medium + 10% DMSO (dimethyl sulfoxide + 30% FBS (fetal bovine serum). The amount of FBS also varied form cells to cells, but a concentration of 30-40% is usually preferred.

5. Re-suspend the pellet with the desired volume of the freezing medium.

6. Aliquot these to each cryovials. Place cryovials directly in dry ice or Mr. Frosty jar. 

7. Place the vials directly in -80 C for at least 4 -6 hours. Cells in this freezer can last up to 1 year.

8. Transfer the vials into the vapor phase or liquid nitrogen storage for long term storage.

THAWING

1. Prior to thawing, pre-warm medium, label the flask and tubes.
2. To bring cells back to life, collect a vial from your cryo storage box or tank.
3. Place the vial in water bath for 1 minute, until no ice is observed in the vial.
4. Take the suspension in the vial and transfer to a new falcon tube. Add 9 ml of warm medium.
5. Centrifuge this suspension at 1500 rpm for 5 minutes.
6. Remove supernatant. Resuspend the pellet with 1 ml medium.
7. Transfer to the new T75  flask where you will grow the cells.
8. Add 14 ml more medium to the flask. Keep the cells in the incubator and observe their growth before using them for experiment.

I hope you find this cell culture guide helpful. Reach out to me anytime via the comments section below for questions or troubleshooting in any cell related work. I am more than happy to hear from your experience and to discuss the A-Z of cell culture! 👍👍😉